The genetic analysis of tumorigenesis is being carried out with the uniquely useful stably diploid, nontumorigenic CHEF/18 cell line (Chinese hamster embryo fibroblast) which was developed in this laboratory. We previously have shown that CHEF/18 cells do not form tumors after single-step mutagenesis, requiring multiple genetic changes to become tumorigenic. We have shown further that CHEF cells are excellent recipients of transfected plasmid or genomic DNAs derived from tumors, and give rise to foci containing tumorigenic cells. This system will be used to determine how many different genes are involved in the tumorigenic transformation of this single cell type, which forms spindle-cell sarcomas in nude mice. For this purpose CHEF/18 cells as well as transformed but nontumorigenic mutants will be transfected with DNA from many different tumors. We have already recovered tumor-derived CHEF cells following transfection with DNA from the human bladder tumor EJ. The tumor-derived cells show rearrangements of EJ DNA and of chromosomes suggesting multiple changes in tumorigenesis. Thus, successful tumorigenic transformation by EJ DNA does not rule out the occurrence of further genetic changes following transfection. In a related study, we have found that CHEF/18 cells can be made tumorigenic by a single treatment with 5-azacytidine, a drug that induces decreased DNA methylation. The molecular basis of this effect and its relation to chromosome changes are under investigation.